Synthesis of β-Maltooligosaccharides of Glycitein and Daidzein and their Anti-Oxidant and Anti-Allergic Activities

The production of β-maltooligosaccharides of glycitein and daidzein using Lactobacillus delbrueckii and cyclodextrin glucanotransferase (CGTase) as biocatalysts was investigated. The cells of L. delbrueckii glucosylated glycitein and daidzein to give their corresponding 4'- and 7-O-β-glucosides. The β-glucosides of glycitein and daidzein were converted into the corresponding β-maltooligosides by CGTase. The 7-O-β-glucosides of glycitein and daidzein and 7-O-β-maltoside of glycitein showed inhibitory effects on IgE antibody production. On the other hand, β-glucosides of glycitein and daidzein exerted 2,2-diphenyl-1-picrylhydrazyl (DPPH) free-radical scavenging activity and supeoxide-radical scavenging activity.


Introduction
Glycitein and daidzein are important and bioactive isoflavones isolated from soybeans whose pharmacological properties such as anticancer, anti-inflammatory, neuroprotective, anticarcinogenic OPEN ACCESS effects, and protective effects against bone loss, hormone-dependent and -independent cancers, cardiovascular diseases, and autoimmune diseases and have been widely studied [1][2][3][4][5][6][7][8][9][10][11]. Despite these pharmacological activities, their use as medicines and functional food-ingredients is limited, because they are scarcely soluble in aqueous solution and poorly absorbed through oral administration.
Glycosylation is an important method for the conversion of water-insoluble and unstable organic compounds into the corresponding water-soluble and chemically stable derivatives. Mizukami et al. reported that glucosyl conjugation was far more effective than cyclodextrin complexation at enhancing the water solubility of hydrophobic compounds such as curcumin [12]. Recently, absorption efficiency of a lipophilic flavonoid, i.e., quercetin, has been reported to be much improved, when converted into its glycoconjugates [13,14]. Glycosylations of glycitein and daidzein are of importance from the viewpoint of pharmacological development of soy isoflavones. We report here the synthesis of βmaltooligosaccharides of glycitein and daidzein by sequential glycosylation with Lactobacillus delbrueckii and cyclodextrin glucanotransferase (CGTase). We also report their inhibitory activity for IgE antibody formation, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, and supeoxide-radical scavenging activity.

Anti-allergic activity of β-glycosides of glycitein and daidzein
The effects of glycitein β-glycosides 2-7 on IgE antibody formation were investigated by an in vivo bioassay using 7S-globulin from soybean as an antigen [17]. The average rat plasma IgE level after treatment of 7S-globulin with or without test compounds was examined. As shown in Table 1 (Table 1). It has been reported that tocopheryl β-glycosides showed inhibitory effects on IgE antibody formation [18,19]. Recently, we reported that 7-O-β-glycosides of genistein and quercetin showed antiallergic activities, whereas the β-glycosides whose sugar is attached at other phenolic hydroxyl groups, exhibited no anti-allergic actions [20]. These findings suggested that the C-7 β-glucoside and β-maltoside of glycitein and/or daidzein did not attenuate the anti-allergic activity, and that phenolic hydroxyl groups at the 4'-position might be necessary for glycosides of glycitein and daidzein to act as anti-allergic species. Studies on the anti-allergic mechanism(s) of the β-glycosides of glycitein and daidzein synthesized here are now in progress.

Anti-oxidant activity of β-glycosides of glycitein and daidzein
The antioxidative activities of glycitein β-glycosides 2-7 and daidzein β-glycosides 9-14 were determined by an in vitro bioassay of their DPPH radical scavenging activity. The antioxidant activities were expressed as IC 50 values and are summarized in Table 2 (10) showed DPPH free-radical scavenging activity, whereas the β-maltosides and β-maltotriosides of glycitein and daidzein 4-7 and 11-14 had no antioxidant activity. The results obtained here suggested that monoglucosides of glycitein and daidzein might be useful free-radical scavenging antioxidants with high aqueous-solubility.
The superoxide-radical scavenging activity of glycitein β-glycosides 2-7 and daidzein β-glycosides 9-14 were expressed as IC 50 values, summarized in Table 2 (9) showed superoxide-radical scavenging activity. The results obtained here suggested that monoglucosides of glycitein and daidzein could be potential superoxide-radical scavenging antioxidants.

Bacterial strain and culture conditions
Culture medium used for growth of L. delbrueckii subsp. bulgaricus (Okayama University of Science) had the following composition (in grams per liter): 20 g of lactose, 5 g of yeast nitrogen base, 20 g of bacto casitone, 1 g of sorbitan monooleate, 2 g of ammonium citrate, 5 g of sodium acetate, 2 g of K 2 HPO 4 , 0.05 g of MnSO 4 , 0.1 g of MgSO 4 . The cells were grown in the culture medium with continuous shaking on a rotary shaker (120 rpm) at 30 °C.

Production of β-glucosides of glycitein and daidzein by L. delbrueckii
The cultures of L. delbrueckii were grown in 500 mL conical flasks containing 200 mL of culture medium at 30 °C. Prior to use for the experiments, the cells were harvested by centrifugation at 8,000 g for 15 min. The β-glucosides of glycitein and daidzein were prepared as follows: substrate (0.2 mmol/flask, 2 mmol total) was added to ten 300 mL conical flasks containing L. delbrueckii cells (5 g) and glucose (1 g) in freshly prepared culture medium (100 mL). The mixture was incubated with continuous shaking on a rotary shaker (120 rpm) for 5 days at 30 °C. The reaction mixture was centrifuged at 8,000 g for 15 min to remove the cells and the supernatant was extracted with n-butanol. The n-butanol fraction was purified by preparative HPLC on YMC-Pack R&D ODS column to give the β-glucoside products.

Production of β-maltooligosides of glycitein and daidzein by CGTase
To a solution containing β-glucosides of glycitein or daidzein (0.1 mmol) and starch (5 g) in sodium phosphate buffer (25 mM, pH 7.0) was added CGTase (100 U). The reaction mixture was stirred at 40 °C for 24 h, and then the mixture was centrifuged at 3,000 g for 10 min. The supernatant was subjected on to a Sephadex G-25 column equilibrated with water to remove CGTase. The fractions containing glycosides were purified by preparative HPLC on YMC-Pack R&D ODS column to give the β-maltooligoside products. Spectral data of new compounds are as follows: used as a positive control. After 20 min at 25 °C, the absorbance was measured at 517 nm. The percentage reduction of the initial DPPH adsorption, i.e., the free-radical scavenging activity, was calculated as follows: E = [(A c − A t )/A c ] × 100, where A t and A c are the respective absorbance at 517 nm of sample solutions with and without the test compounds. Antioxidant activity was expressed as the 50% inhibitory concentration (IC 50 ).

Superoxide-radical scavenging activity
Superoxide was generated by the xanthine-xanthine oxidase system [21]. Reaction mixture contained 4 mM xanthine (50 μL), various concentration of sample in ethanol (50 μL), 2 mM nitro blue tetrazolium (NBT, 50 μL), of 0.3 nkat/mL xanthine oxidase (50 μL) and 0.1 M phosphate buffer (pH 7.4) in a total volume of 2 mL. Vitamin C was used as a positive control. The reaction mixture was incubated at 25 °C for 10 min and the absorbance was read at 560 nm. Percent inhibition was calculated by comparing with control without test compound but containing the same amount of alcohol. The IC 50 value is shown as the sample concentration at which 50% of superoxideradical was scavenged.

Conclusions
The β-maltooligosaccharides of glycitein and daidzein were successfully produced through two-step biocatalytic glycosylation by L. delbrueckii and CGTase. The 7-O-β-glucoside of glycitein and daidzein and 7-O-β-maltoside of glycitein inhibited IgE antibody formation. On the other hand, β-glucosides of glycitein and daidzein exerted DPPH free-radical scavenging activity and supeoxideradical scaventing activity.